Morphology of the anther, microsporogenesis and pollen structure of Momordica balsamina

Morphology of the anther, microsporogenesis and pollen structure of Momordica balsamina Helene Janse van Rensburg, P.J. Robbertse and J.G.C. Small Margaretha Mes Institute for Seed Research, Department of Botany, University of Pretoria, Pretoria

The morphology of the S-shaped anthers of Momordica balsamina L. corresponds to those of other representatives of the family Cucurbitaceae. A brief discussion of the secretory trichomes present on the anther connective, as well as the actual number of stamens as based on the filament vasculature, is given. During microsporogenesis 'ext ranuclear nucleoli ' were observed in the microspore mother cells. The pollen grains are sub-spherical , tri· colporate with a reticulate tectum and are discharged during the two-celled stage. S. Afr. J. Bot. 1985, 51: 125-132 Die morfologie van die S-vormige helmknoppe van Momordica balsamina L. stem ooreen met die van ander verteenwoordigers van die familie Cucurbitaceae. 'n Kort bespreking van die sekreterende trigome wat op die helmbindsel voorkom, sowel as die ware getal meeldrade soos gebaseer op die bearing van die helmdraad , word gegee. Tydens mikrosporogenese is 'ekstranukluere nukleolusse' in die mikrospoormoederselle waargeneem . Die stuifmeelkorrels is sub-sferies, trikolporaat met 'n geretikuleerde tektum en word gedurende die tweesellige stadium vrygestel. S.·Afr. Tydskr. Plantk. 1985, 51: 125-132

Keywords: Filament vasculature, microsporogenesis, Momordica, pollen, secretory trichomes

Introduction Momordica ba/samina L is a member of the family Cucurbitaceae. It is indigenous to southern Africa and occurs in Botswana, South West Africa/ Namibia as well as in the Republic of South Africa (Meeuse 1962). The plants occur abundantly in the tropics and subtropics (Trivedi & Roy 1972) in sand veld. M. balsamina is a monoecious, herbaceous climber with ephemeral aerial parts and subterranean parts which may be perennial. According to Watt & Breyer-Brandwijk (1962), this plant has medicinal properties and is used by local African tribes as a cathartic and also for curing haemorrhoids and prolapsus ani. From the literature available, it is clear that no concensus of opinion regarding the basic number of stamens found in the Cucurbitaceae has been reached; no detailed description of the ultrastructure of the pollen exists and very little information is available on the structure of the unusual trichomes on the anthers. This study was undertaken to fill this gap in our knowledge of the Cucurbitaceae and forms part of investigations undertaken at the Margaretha Mes Institute for Seed Research concerning endangered, rare and economically important indigenous South African plants. Materials and Methods

Helene Janse van Rensburg* Present address: Ontario 603, 579 Adcock Str. Gezina, Pretoria, 0084 Republic of South Africa P.J. Robbertse and J.G.C. Small Margaretha Mes Institute for Seed Research, Department of Botany, University of Pretoria, Pretoria, 0002 Republic of South Africa *To whom correspondence should be addressed Accepted 26 November 1984

Plants were cultivated in the garden of the University of Pretoria. Flowers in various stages of development were collected and fixed either in FAA or 60?o glutaraldehyde. Flowers used in S.E.M. studies were dehydrated in an ethanol series, critical-point dried and then sputter-coated with gold prior to viewing with a Phillips S.E .M. 500 scanning electron microscope. Pollen grains were acetolysed according to the method of Coetzee & Vander Schijff (1979), sputter-coated with gold and viewed with the same microscope. Material for semi-thin sections was dehydrated with 100% ethyleneglycolmonomethylether, 100% ethanol, 100% butanol, 100% propan-1-ol and embedded in glycomethacrylate (G.M.A.) as prescribed by Feder & O'Brien (1968). Sections, ca. 2 11m thick, were made with glass knives on a Reichert O.M.U. 3 ultra-microtome. The periodic acid/Schijffs reaction (P.A.S.) (O'Brien & McCully 1981) was used, with 0,5% 2,4 dinitrophenyl hydrazine (D.N.P.H.) in 15% acetic acid as a blocking agent. Sections were counterstained for 1 - 5 min in 0,05% toluidine blue solution dissolved in a benzoate buffer at pH 4,4 (Sidman, Mottla & Feder 1961). Material for thin sections was fixed in 6% glutaraldehyde and post-fixed in 2% Os04. It was then dehydrated in an

126

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Figures 1- 4 (1) Stamen with multicellular trichomes (Mt). A - anther; F - filament. Scale line = 500 ~m. (2) Diagram of multicellular trichomes in longitudinal section . Be - basal cell; Tc - terminal cells. Scale line = 20 ~m. (3) Mature S-shaped anther (A). F - filament. Scale line = 200 ~m . (4) Dehiscing anther. P - pollen grains; T - trichomes. Scale line = 100 ~m.

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ethanol series followed by a 1,2 propylene oxide and embedded in Spurr's resin (Spurr 1969). Sections ca. 100 nm thick were made with a diamond knife on a Reichert O .M.U. 3 ultramicrotome. These were stained in 50?o uranylacetate and lead citrate (Reynolds 1963) and viewed with a Phillips E.M. 301 transmission electron microscope. To study meiosis, young male flower buds were collected every half hour from 07h30 to 14h30 and fixed for 6 - 12 h

in Pienaar's reagent (Johansen 1940). Peacock's (1966) propioncarrnine squash technique was used. Callose walls were identified by mounting G.M .A. sections in 0,1 O?o aniline blue in 0,3 mol dm - 3 K3P04 buffer. These sections were examined with a Reichert UNIV AR microscope with epifluorescent optics. The vasculature of the filaments as well as whole mounts of trichomes on the stamens were studied by means of a

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Figures 5 - 8 (5) Paired anthers (A) with vascular bundles (Vs). Scale line = 500 ~m. (6) Single anther (A) wi th vascular bundle (Vs). Scale line = 500 ~m. (7) Female flower with petals removed to show the staminodia (St) . 0 - ovary; Se - sepal; Sg - stigma. Scale line = 600 ~m. (8) Transverse section of the upper part of the female flower. Pe - petal; S - style; Se - sepal; St - staminodia. Scale line = 100 ~m.

128

5. -Afr. Tyds kr. P lantk ., 1985, 5 1(2)

clearing-squash technique (Herr 1971) on material stained with 0,1 OJo Sudan IV in 100% ethanol. To describe the pollen, the terminology of Erdtman (1969) and Kapp (I 969) is used. Results

Stamen morphology Phenetically, the androecium of Momordica balsamina, like other Cucurbitaceae species described by Eichler (1954), consists of three epipetalous filaments which are free at their bases but fused terminally. Two of the filaments each have two S-shaped, bisporangiate anthers simulating a tetra-

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sporangiate stamen (Figures I & 3) and giving the impression of tetrasporangiate stamens. The remaining filament has one S-shaped, bisporangiate anther. S-shaped, monothecous, bisporangiate anthers are typical of the Cucurbitaceae. The same androecium configuration was described by Heimlich (I 927) for the flower of the cucumber and by Zimmermann (1922) for the genus Momordica, and corresponds to the female flower where two of the staminodes are bilobed and one is unilobed (Figures 7 & 8). A closer examination of the cleared filaments, however, showed two vascular strands in each of the two 'tetrasporangiate' stamens and only one vascular strand in the stamen with one bisporangiate anther

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Figures 9 - 13 (9) Pa rt of mature anther wall and microspores (Mi). En - endothecium; Ep - epiderm; Om - outer middle layer; Wt - wall thickenings. Scale line = 30 Jlm. (10) Endothecium with wall thickenings (Wt). X 1069. (11) P art of an anther wa ll. E n endothecium; Ep - epidermis; Im - inner middle layer; Mi - micros pore; Om - outer middle layer; T a - tapetum. x 2274. (12) Part of an anther with stomium cells (Sc), common microsporangium wall (Mw) and pollen grains (P). Scale line = 30 Jlm. (13) Mature anther with microspores (Mi), stomium cells (Sc) at the apex and a degenerating common microsporangium wall (Mw). Scale line = 30 11m.

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S. Afr. J. Bot., 1985, 51(2)

(Figures 5 & 6). The basal parts of the filaments are covered with multicellular trichomes (Figure 1). A different type of trichome (Figures 2 & 4) occurs at the basal part of the connective and also between the folds of the thecae (Figures 4 & 14). The latter trichomes consist of a large basal cell and three to eight smaller terminal cells (Figure 2). The terminal cells are shortlived and are shed at the time of pollen liberation. At the same time the contents of the basal cell are secreted by the shrinking of the plasmalemma. The terminal cells have thin, cellulosic walls, while the outer part of the thick walls of the basal cells and adjacent epidermal cells are impregnated with an osmophylic substance (Figure 15). The lipoid nature of these walls was demonstrated by staining the trichomes with Sudan IV solution. Lipid bodies, plastids, numerous mitochondria and vacuoles of different sizes are apparent in the cytoplasm of the basal cells (Figure 15).

Ontogeny of the stamens The stamina! primordium consists of a mass of homogeneous meristematic cells covered by a protoderm. These cells differentiate into the anther wall and the microspore mother cells which eventually form the microspores. The young anther wall consists of an epidermis, an endothecium, two middle cell layers and a tapetum (Figure 16), thus conforming to the Basic type (Davis 1966). The hypodermal endothecium is well defined and reaches its maximum differentiation just before the pollen is shed (Figures 9, 10 & II). Secondary cell wall thickenings (Figures 9 & 10), which are not visible during meiosis and which are related to the anthers' dehiscence (Varghese & Grover 1972), occur at this stage. The tapetum is of the secretory type (Foster & Gifford 1974) since the cell walls remain intact until after meiosis (Figure 19). In spite of their larger vacuoles the tapetum cells are more electron dense than the other wall layers. The inner middle

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Figures 14- 17 (14) Transverse section of an anther (A). Be - basal cell; En - endothecium; Ep - epiderm; Mi - micros pores; Tc - terminal cells . Scale line = 100 Jlm. (15) Section through a multicellular hair at the base of the stamen. Ep- epidermis; 01 - osmiophylic layer. x 1348. (16) Transverse section through part of a young anther. En - endothecium; Ep - epidermis; M - middle layers; Mm - microspore mother cells; Ta - tapetum. Scale line = 40 Jlm. (17) Microspore mother cells (Mm) at the beginning of meiosis. Pe - petal. Scale line = 200 J.!m.

130 cell layer disintegrates more or less at the same stage as the tapetum cells, whereas the outer middle layer is persistent (Figures 12 & 13). Dehiscence of the anthers occurs by means of longitudinal

S. -Afr. Tydskr. Plantk., 1985, 51(2)

slits (Figures 1 & 3) which form at the junction between the microsporangia (Figures 12, 13 & 14). At the same time, the common microsporangium wall of each anther degenerates (Figure 14).

Figures 18 - 23 (18) Fluorescent callose wall (C) surrounding the microspore mother cells (Mm). Ta - tapetum. Scale line = 20 J.!m. (19) Anther with tetrads (Te). En - endothecium; Ep - epidermis; Im - inner middle layer; Om - outer middle layer; Ta - tapetum. Scale line = 30 J.!m . (20) Meiosis stage with extranuclear nucleoli (E). Scale line = 10 J.!m. (21) Anaphase 1: Ch - chromosomes. Scale

line = 10 J.!m. (22) Cytokinesis during microsporogenesis resulting in formation of tetrads. Cw - cell wall. Scale line = 10 J.!m .(23) Transverse section of a mature pollen grain. A - aperture; Ex - exine; Gc - generative cell; I - intine; N - nucleus; Nu - nucleolus; PI - plasmamembrane; Va - vacuole; Ve - vegetative cell . x 1568.

S. Afr. J . Bot., 1985, 51(2)

Microsporogenesis and pollen structure The microspore mother cells developing from the sporogenous cells are enclosed in callose walls (Figures 17 & 18). Meiosis occurred between 09h00 and !OhOO and lead to the formation of tetrahedral tetrads (Figure 19). Tetrad formation results from simultaneous cytokinesis. During the prophase stage of meiosis, darkly stained bodies were observed in the microspore mother cells (Figure 20). They correspond to the 'extranuclear nucleoli' observed in Micrampe/is /obata (Kirkwood 1907) and which were later also found in Cucurbita pepo (Passmore 1930). According to Kirkwood (1907), these bodies originate from cytoplasmic fibres appearing during the growth of the microspore mother cells. Prior to the tetrad stage these bodies are distributed in the cytoplasm, but at the conclusion of meiosis, they become grouped around the nuclei and are distributed among the microspores (Figure 22). These bodies disappear in the maturing microspores. Eleven bivalent chromosomes were counted during metaphase I (Figure 21). This confirms the number recorded by Whitaker (1933) for Momordica. The pollen grains of M. ba/samina are liberated in the two-

131 celled condition (Figures 12 & 23). The mitotic division to form these cells is unequal and results in a small generative and a larger vegetative cell (Figure 23). These cells are separated by a plasmamembrane. The cytoplasm of the mature pollen grains contains many vacuoles, mitochondria, lipids and a few Golgi-bodies, but no starch grains were observed. The pollen grains are oblate spherical (P = 4 j..t.m; E = 50 j..t.m; PIE = 0,92), tricolporate with evenly spaced apertures (Figure 24). The surface sculpturing is pertectate reticulate (Figures 24 & 25). The electron dense exine consists of an outer sexine with tectum, bacula and floor layer and an inner nexine (Figure 26). The intine is less electron dense than the exine but has about the same thickness. In the mature pollen grains of other Cucurbitaceae taxa, Kirkwood (1907) found the exine to be thinnest in the vicinity of the pores. Our results show a similar situation in M. ba/samina. Futhermore, it was observed that the intine is very thick in in this region (Figures 23 & 27).

Discussion The morphology and anatomy of the male flower of

Figure~ 24 - 27 (~4) Aceto lysed pollen grain. A - aperture; Ap - apocolpium; Co - colpium; I - intine; Me _ mesocolpium; Po - por~um. Sca~e !me = 10 ~m. (2.5) Surface struc~ure of an acetolysed pollen grain. Scale line = 1 ~m. (26) Pollen grain wall. Ba _ ba.cula, I - . mtme; L - lacuna; LI- lamellar mtme; N- nexine; Tt- tectum. x 13720. (27) Edge of pore. Cy _ cyto Jasm· E _ exme; I - mtme. x 3972. p '

132 Momordica ba/samina are similar to those of other members of the family Cucurbitaceae as described by Eichler (1954). From the literature available it is clear that there is no concensus of opinion regarding the basic number of stamens found in the Cucurbitaceae. According to Chakravarty (1958), Bentham & Hooker, Clarke, Britton & Brown, Small, Ridley & Gamble reported the presence of three stamens, while Gray & Heimlich found only two and a half stamens. Chakravarty (1958), however, concluded that the basic number is five and that each stamen has only one vascular strand. Thus, although the stamens are monothecal, they should not be regarded as half stamens but as complete stamens which have lost one theca. This viewpoint supports that of Zimmermann (1922) and is also in accordance with the condition found in M. ba/samina. In M. balsamina the apparently trimerous androecium consists of two pairs of adnate, but completely monothecous stamens, and one separate monothecous stamen. Trichomes, at the base of the connective and between the folds of the theca as found here in M. ba/samina, have also been investigated by Zimmermann (1922) in several other species of the family Cucurbitaceae. He referred to these trichomes as 'klebstofhaare' which produce oil that makes the pollen grains sticky. The chemical nature and possible function of the trichomes need to be investigated in more detail. The microspore mother cells of M. ba/samina each possess 11 chromosome bivalents. This confirms the reports of Whitaker (1933) and Trivedi & Roy (1972). The only other Momordica species of which the chromosome numbers have been determined are Momordica dioica (14 bivalents) and Momordica charantia (II bivalents) (Trivedi & Roy 1972). Passmore (1930) commented on the paucity of literature concerning the development of the microspore mother cells of representatives of the Cucurbitaceae. According to Kirkwood (1907), who investigated the pollen of various members of the Cucurbitaceae, the exine in the mature pollen grains is thinnest at the aperture. The intine of the pollen of M. balsamina is relatively thick at the aperture. No literature concerning the pollen wall ultrastructure of species of the Cucurbitaceae could be traced. This study therefore represents the first detailed investigation of the ultrastructure of the pollen grain wall. The pollen grains of M. balsamina are liberated during the two-celled stage. This is in accordance with the observations of Kirkwood (1907) on other representatives of the Cucurbitaceae. Conclusion

Although the androecium of M. ba/samina consists of three filaments, it is in fact pentamerous. The five vascular strands occur in a (2) + (2) + 1 arrangement. Trichomes of unusual structure occur on the anther connective. The chemical nature and possible function of these trichomes should be investigated in more detail. Detailed information of the ultrastructure of the pollen grain wall is supplied. Acknowledgements

The research was supported in part by research grants from the South African C.S.I.R. and the University of Pretoria. We thank Miss H . Coetzee for her critical reading of the . manuscript and Mr C.F. van der Merwe for the electronmicrographs.

S.-Afr. Tydskr. Plantk ., 1985, 51(2)

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